JP6635383B2 - Graphene and graphene-related materials for manipulation of cell membrane potential - Google Patents

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from US Provisional Patent Application No. 61 / 998,089, filed June 17, 2014, which is hereby incorporated by reference in its entirety.

  Changes in cellular membrane potential are based on a number of basic physiological processes, including heart rate, brain function, visual and olfactory transmission, and muscle contraction. Cells often use electrical signaling to communicate with each other and to change their behavior according to external cues.

  The plasma membrane ensures the integrity of the cell's structure and physically separates the intracellular compartment from the extracellular surroundings. Due to the substantial differences in ionic composition between the intracellular and extracellular compartments and b) the physical properties of the insulating properties of the phospholipid bilayer containing the cell membrane, an electric field (E-field) ) A gradient exists. At rest, the inside of the cell is more negatively charged than the outside, resulting in a membrane potential in the range of -10 to -80 mV for various types of cells. Changes in membrane potential can be caused by electrical and / or chemical signals. Specifically, these signals lead to the opening of voltage-gated or ligand-gated ion channels, resulting in altered membrane permeability, redistribution of ions inside and outside the plasma membrane, and finally The membrane potential changes.

  To regulate the membrane potential is to regulate the activation state of the cell. Changes in membrane potential are linked to fundamental functions such as cellular excitability, post-translational modification of proteins by voltage-dependent phosphatases, hormone secretion, ion homeostasis, regulation of protein expression and cell growth, and therefore have various downstream Can lead to the effect of A special situation exists in excitable cells (eg, neurons or cardiomyocytes (CM)) where relatively small changes in membrane potential are amplified, resulting in a coordinated multiple ion channel-type cascade. Activation can lead to the generation of action potentials. Even in non-excitable cells, changes in membrane potential result in changes in electrochemical ion gradients and ion concentrations, which can trigger many apparently voltage-dependent processes.

  Many in vitro applications that require sophisticated methods for manipulating cell membrane potential include basic research on molecular mechanisms in health and disease; functional search for voltage-sensitive membrane proteins; information exchange between cells including synaptic plasticity Studies; heart disease-related projects; visual and olfactory transmission pathways; generation and characterization of stem cell-derived cells for cell replacement therapy; activation-dependent maturation and differentiation of stem cell-derived cells; and their potential cardiotoxicity High-throughput screening of candidate drugs to perform and support the development of personalized medicine.

  The novel methods for manipulating cell membrane potential will also be very useful for in vivo applications in situations where it is necessary to enhance endogenous activation triggers that are impaired during disease progression. For example, modulation of the overall membrane potential of the CM can help overcome incomplete electrical signaling and thus regulate abnormal heart rhythms. Photoreceptor degeneration during retinitis pigmentosa or age-related macular degeneration can be offset via light-driven electrical stimulation of the next row of intact cells in the retina, potentially leading to visual recovery There is. Stimulation of neurons can help offset neuronal deficits in patients with cerebrovascular, neurodegenerative and psychiatric disorders.

  Electrophysiology is the most direct and accurate way to regulate cell membrane potential. In general, electrophysiological stimulation by electrode-based techniques (eg, patch pipettes, multi-electrode arrays or E-field stimulation) exhibits a high degree of regulation on cell membrane potential. However, these techniques have significant disadvantages, such as limited spatial selectivity, invasiveness, low throughput, lack of automation, and the overall mechanical stiffness of modern multi-electrode materials. In addition, electrode-based techniques are less suitable for studies in which CMs contract because of the damage that stationary electrodes cause in cells exhibiting normal physiology.

Chemical methods for inducing changes in membrane potential utilize approaches such as the application of “high K ⁺ ” extracellular solutions or pharmacological ion channel openers. Significant disadvantages of this method include the inability to modulate the membrane potential, the irreversibility of the induced changes and poor temporal and spatial resolution.

  Optical stimulation methods utilize light beams as actuators and may therefore allow for long-term, non-invasive regulation of cell membrane potential. However, existing methods have serious inherent disadvantages. One of the oldest methods in this category is the light-induced "release" of chemically modified signaling molecules such as glutamate, ATP, dopamine, serotonin, cyclic nucleotides, and the like. Although this method has a high temporal resolution, its spatial resolution is limited due to diffusion of the "released" molecules from the release site. In addition, "released" chemicals exhibit precursor instability, phototoxicity, and require strong UV irradiation. Furthermore, the "release" process is irreversible and non-repetitive.

  Optogenetics, the latest and most promising method of optical stimulation, is to use transmembrane proteins encoded by genes whose sensitivity to light is either intrinsic or altered. Optogenetics allow for the regulation of membrane potential in selected populations of cells with unmatched spatiotemporal accuracy. However, to effect light-mediated regulation, optogenetics requires the expression of exogenous, functionally active proteins that are an integral part of the intracellular machinery. In other words, researchers must alter cell physiology to be able to regulate cell behavior, which can hinder research using stem cell-derived cells. Some of the technical challenges of optogenetics have already been addressed during the multi-iterative process of protein engineering and the development of large-scale tools over the last few years. However, some problems are inherent in this approach: 1) optogenetics is not physiological because ionic flux and initial membrane potential changes are determined by properties of foreign proteins rather than endogenous ion channels. Does not provide irritation. 2) High expression levels of the photosensitive protein are required to achieve the desired change in membrane potential. 3) In many cases, the photosensitive function of optogenetic proteins requires the addition of the endogenous cofactor all-trans retinal or a photocatabolic compound. 4) Optogenetics is not suitable for pharmacological profiling of new drugs because new drugs can directly affect opsin instead of or in addition to the target of interest.

  Materials Science: Using substrates composed of inorganic bulk semiconductors, semiconductor nanoparticles, and organic semiconductor polymers, the light-induced electrical excitation of neurons has been revealed. For example, neurons cultured on silicon wafers can be activated by light, which is a light-induced change in silicon conductivity in the presence of a specific potential applied across the wafer. Can cause a capacitive transient response in neurons, resulting in depolarization of their membranes. Unfortunately, 1) this platform is not transparent, and thus has limited compatibility with optical detection methods; 2) its applicability due to the very expensive and very strong silicon wafers in the elaborate adjustment device. Severely limited; 3) The spatial resolution defined by the photocurrent expansion diameter is ≧ 50 μm. Some of these issues were addressed in substrates where the photosensitive layer was made from a mixture of organic semiconducting polymers. Compared to a silicon-based substrate, this platform does not require any external equipment, has low heat dissipation, is flexible, and is very easy to fabricate. Disadvantages include: a) incompatibility with optical detection due to low transmission and excitation in the visible range; and b) possible miniaturization problems due to mechanical fragility of the polymer layer. . Another optical stimulation platform based on semiconductor nanoparticles had the greatest engineering flexibility during its fabrication, but was poorly biocompatible due to the presence of cadmium-containing components in the nanoparticles.

  In summary, new technical tools are needed for remote stimulation of cells. The optimal physiological stimulation tool is to adjust the width, duration and direction of these changes; to quickly and reversibly increase the membrane potential, so as to be less invasive and compatible with non-invasive detection methods. It should be possible to change iteratively.

To meet these requirements, new materials with appropriate properties can be considered. A family of graphene-based or graphene-related materials (where the terms "graphene-based" and "graphene-related" are used interchangeably) has recently gained attention after the 2010 Nobel Prize in Physics, The development of applications for these materials in the fields of energy, electronics, sensors, light processing, medicine and the environment has increased exponentially. Graphene is a member "from the beginning" of the family, sp 2 arranged in a hexagonal honeycomb grating ^- is a two-dimensional material made from a hybrid carbon atoms. The broad family of graphene-related materials includes graphene (single and multi-layer), graphite, polycyclic aromatic hydrocarbons, carbon nanotubes, fullerenes, various graphene nanostructures of various dimensions (eg, graphene nanoparticles or graphene quantum Dot: graphene nanoribbon: graphene nanomesh; graphene nanodisk; graphene foam; graphene nanopillar), other graphene-related materials, substituted graphene-related materials (eg, replacement of carbon atoms with N, B, P, S, Si, etc.) and , Reactive functional groups (for example, carboxyl group, ester, amide, thiol, hydroxyl group, diol group, ketone group, sulfonic acid group, carbonyl group, aryl group, epoxy group, phenol group, phosphonic acid, amine group, porphyrin, Pyridine Any combination of graphene-related materials which are functionalized with a polymer, and combinations thereof). Specific examples of the graphene-related material include graphene oxide (GO), graphite oxide, and reduced graphene oxide (rGO).

  Graphene and graphene-related materials exhibit phenomenal electronic, mechanical and optical properties, which allow graphene and graphene-related materials to construct biocompatible interfaces for remote stimulation of cells using electromagnetic radiation And can be valuable in a variety of biomedical applications, including: Graphene is very inert and chemically stable, and therefore has excellent biocompatibility. Its unique properties include high electrical conductivity, high charge carrier mobility, very good mechanical properties (eg, high rigidity, strength and elasticity), high thermal conductivity, broadband absorption and high transmission for the visible spectrum Sex. Further, by performing one of many routes, such as changing the stacking pattern of the graphene sheet, changing the shape of the graphene structure, replacing carbon atoms in the graphene lattice, and functionalizing the graphene, it can be used in specific applications. In contrast, it is possible to adjust the electronic and optical properties of graphene.

Graphene has a zero bandgap, which has led to broadband absorption of incident light at energies below about 3.5 eV. This means that graphene with a high light absorption coefficient (7 × 10 ⁵ cm ⁻¹ ) can efficiently detect wavelengths of light in the range of 300 to 2500 nm, including the visible spectrum, infrared and even the entire terahertz range. Means

The graphene monolayer is very transparent, with an absorption of 2.3% for the visible spectrum and an absorption peak of about 10% for UV. When graphene absorbs photons, it converts their energy to electrical current by causing photo-generated excitation via a photoelectric and / or photo-thermoelectric mechanism. The photoresponsiveness of graphene is somewhat due to the lower light absorption in single-layer graphene, the shorter recombination lifetime (on the picosecond scale) of photogenerated carriers, and a lower internal quantum efficiency of about 6-16%. Low (<10 mAW ⁻¹ ).

  GO is a graphene-related material that is highly oxidized with many oxygen-containing functional groups. In contrast to graphene, which has its zero bandgap and high electronic conductivity, GO is a wide bandgap semiconductor with a bandgap> 3.5 eV and very poor electronic conductivity. Removal of the oxygen-containing functional group reduces the optical band gap from> 3.5 eV to <1 eV, increases light absorption, and restores electrical conductivity. The result of this degradation process is rGO or another graphene-related material known as chemically converted graphene. While graphene and other non-functional graphene-related materials (eg, graphite, carbon nanotubes and fullerenes) are hydrophobic, GO is hydrophilic due to the presence of oxygen-containing functional groups. rGO is moderately hydrophilic because the number of residual oxygen-containing functional groups in rGO is less than in highly oxidized GO.

  Among the latest biomedical applications for graphene and its derivatives are their use for tissue engineering, antimicrobial treatment, drug and gene delivery, and as contrast agents for bioimaging. For example, the incorporation of graphene-related materials as structural components, both into planar or three-dimensional scaffolds for cell culture, greatly enhances cell adhesion, improves cell growth, accelerates cell maturation rates, It promoted process sprouting and elongation and supported neural cell lines undergoing stem cell differentiation. These applications have not only utilized pure graphene and its derivatives, but also their hybrid nanocomposites (eg, semiconductor quantum dots, carbon nanotubes), proteins (eg, chitosan), polymers (eg, poly) (Propylene carbonate)) or other chemicals (eg, PEG) were also utilized. It has been suggested that the favorable effects of graphene-related materials in creating a favorable cellular microenvironment are based on their mechanical, microscale topographic and surface chemistry characteristics.

  All existing biomedical applications make use of the passive steady-state properties of graphene and its derivatives. Currently, there are no applications that utilize external stimuli to actively change the physicochemical properties of graphene-related biointerfaces and subsequently to change the functional state of cells that interact with these interfaces.

  Disclosed is the use of a biocompatible interface that includes a graphene-related material (G-biointerface) for manipulating the electrical properties of cell membranes, both in vitro and in vivo.

  In certain embodiments, a biocompatible interface can include one or more structures of a graphene-related material. In another embodiment, one or more chemicals may be added to, functionalized with, and / or added to the graphene-related material. In certain embodiments, the G-bio interface may be utilized as a stand-alone structure. In other embodiments, a G-biointerface structure may be deposited on a substrate.

  In a further embodiment, a method of manipulating membrane potential via a G-biointerface comprises: positioning a biological target near the G-biointerface; and exposing the G-biointerface to an electromagnetic stimulus. May be included. In certain embodiments, the electromagnetic stimulus can be a visible spectrum electromagnetic stimulus.

  A method of performing a biological assay can include inducing a change in membrane potential via the G-biointerface and monitoring the resulting change using one or more detection methods. In certain embodiments, the detection method is an optical optic that, when combined with a G-interface activated by light to manipulate membrane potential, can result in an all-optical assay probing biological activity. Method. In certain embodiments, the detection method can be a genetic method. In certain embodiments, the detection method can be an electrophysiological method.

  A method of treating an animal having a medical condition can include administering a G-biointerface structure to the animal and exposing the G-biointerface to an electromagnetic stimulus, wherein the medical condition comprises: Parkinson's disease; Amyotrophic lateral sclerosis; Huntington's disease; Chemotherapy-induced neuropathy; Age-related cerebral dysfunction; Down syndrome; Autism spectrum disorder; Cerebral palsy; Epilepsy and other paroxysmal disorders; Disorders, depression, TBI / PTSD / CTE (chronic traumatic encephalopathy) and other stress, traumatic or blast injuries or diseases; schizophrenia and other mental disorders; pain, hyperalgesia and nociceptive disorders; Addiction disorder; nerve ischemia; nerve reperfusion injury; nerve trauma; nerve hemorrhage; nerve damage; nerve infection; stroke; Systemic disorder; arrhythmia; heart failure; cardiomyopathy; rheumatic heart disease; coronary artery disease; congenital heart disease; eye disease; age-related macular degeneration; retinal pigment degeneration; glaucoma; diabetic retinopathy; calcium homeostasis; Producing tumor; diabetes; hypoglycemia; adrenal disorders; thyroid disorders; growth disorders; metabolic bone disorders; sex hormonal disorders;

  For a more complete understanding of the present disclosure and its advantages, reference is now made to the following description, taken in conjunction with the accompanying drawings, which describe specific embodiments of the present disclosure.

1 illustrates a general strategy for utilizing graphene and graphene-related materials in a biocompatible optoelectronic interface to perform remote stimulation of cells using electromagnetic stimulation. 1 shows brightfield optical microscope (A) and scanning electron microscope (B) images of CM on glass coverslips coated with graphene (left) and rGO (right). 9 shows high biocompatibility of substrates based on rGO. Long-term culture of CM in bright field (left) and fluorescence (right) light microscopy images of CM cultured on heterogeneous substrate as marked (top is glass, bottom is rGO coated surface) ). CM is labeled with nuclear dye Hoechst (blue) and cytoplasmic dye calcein (green). (B) Evaluation of cell viability via CM vs. glass surface versus rGO coated surface cultured CM normalized cell density (left) and membrane integrity (right). Cell density was calculated as the number of cells in the field normalized to a control value for cells cultured on glass coverslips. Membrane integrity values were given as the percentage of cells that exhibited a fluorescent signal after staining with calcein-AM and ethidium homodimer-1 (EthD-1). Data are means ± sd. e. m. (N ≧ 100 cells / each condition). The effect of light irradiation (blue bars) on the frequency of spontaneous contractions in CM under various experimental conditions is shown: (A) long-term culture of CM on graphene-coated glass coverslips, (B) on rGO-coated glass coverslips. -Term culture of CM on (C) Short-term contact of rGO slices with CM cultured on "naked" glass coverslips. Brightfield microscopy images were used to create representative traces reflecting changes in optical density near the edges of the cells as a result of CM contraction. Figure 4 shows the effect of light irradiation on the action potential of CM cultured on glass coverslips (A) and rGO coated glass coverslips (B, C). The vertical bar is 10 mV. Shown are individual traces of light-induced changes in intracellular calcium concentration in multiple CMs cultured on rGO coated glass coverslips and labeled with Fluo4, a fluorescent calcium sensitive indicator. Figure 7 shows long-term culture of induced pluripotent stem cell (iPSC) -derived neurons seeded on rGO coated glass coverslips as represented by brightfield light microscopy images (A) and scanning electron microscopy images (B). 9 shows the excellent biocompatibility of rGO coated glass coverslips for iPSC-derived neurons. (A) Neurons cultured on glass coverslips (left) and rGO coated glass coverslips (right) were labeled with calcein-AM and EthD-1 to label live (green) and dead cells (red), respectively. Stained. (B) Summary of cell viability experiments. Data are means ± sd. e. m. (N ≧ 100 cells / each experimental condition). Figure 4 shows the effect of light irradiation on the membrane potential of neurons cultured on rGO coated glass coverslips. (A) Experimental scheme: monitoring activation of neurons using light and ensuring electrical activity using electrophysiological methods. (B) Light-evoked action potential in neurons on rGO coated glass coverslips. Figure 3 shows the photo-induced effect on intracellular calcium ion distribution in neurons cultured on rGO coated glass coverslips. (A) Experimental scheme: activation of neurons using light and monitoring ensuring changes in intracellular calcium concentration using optical methods. (B) Individual traces of light-induced changes in intracellular calcium in neurons cultured on rGO coated coverslips and labeled with Fluo4, a fluorescent calcium indicator.

  The present disclosure is not limited to the particular systems, devices, and methods described, as these may vary. The terms used in the description are intended to describe certain variants or embodiments only, and shall not limit the scope thereof.

  The present disclosure provides, among other things, nanostructured biocompatible switchable interfaces based on graphene-related materials, methods of preparing such biointerfaces, and the use of such biointerfaces for cell generation, verification and characterization Methods of using such biointerfaces for pharmacological profiling of cells and chemicals, and methods of treating animals with medical conditions using such biointerfaces.

  The general design strategy is based on the following sequence of events shown in FIG. 1) During exposure to electromagnetic stimuli, the graphene-related material absorbs its energy, resulting in excitons and subsequent dissociation into electrons and pores. 2) Due to the capacitive coupling effect between the cell membrane and the surface of the graphene-related material, photogenerated free charge carriers (eg, electrons) from the G-bio interface displace cations near the graphene / cell membrane interface. 3) Such redistribution of the charge immediately outside the cell membrane causes a change in cell membrane potential. The large interfacial volume of the G-biointerface provides effective capacitive stimulation. Thus, the interaction of the E-field gradient between the E-field and the membrane caused by extraneous light near the G-biointerface leads to non-invasive remote stimulation of cells.

  The graphene-related material in the G-biointerface may generally be any material derived from graphene or converted to graphene using various methods of fabrication, functionalization or substitution. The family of graphene-related materials includes graphite, graphene, polycyclic aromatic hydrocarbons, substituted graphene-related materials (with some carbon atoms replaced by atoms such as N, B, P, S, Si) and carboxyl Groups, esters, amides, thiols, hydroxyl groups, diol groups, ketone groups, sulfonic acid groups, carbonyl groups, aryl groups, epoxy groups, phenolic groups, phosphonic acids, amine groups, porphyrins, pyridines, polymers and combinations thereof Contains graphene-related materials that are functionalized with functional groups. An example of a functionalized graphene is GO, which contains many oxygen-containing functional groups. Another example of a functional graphene is rGO produced by removal of at least some oxygen-containing functional groups from GO.

  Graphene-related materials can be of any dimension. For graphene, examples of zero-dimensional nanostructures include graphene nanoparticles or graphene quantum dots; examples of one-dimensional nanostructures are graphene nanoribbons; examples of two-dimensional nanostructures are graphene sheets, graphene nanomesh Examples of three-dimensional nanostructures are graphite, a stack of graphene sheets; carbon nanotubes, roll-up sheets of graphene; fullerenes, wrap-up sheets of graphene; graphene foam; and graphene nanopillars.

  Any graphene-related material incorporated into the G-biointerface can be used alone or can be part of a hybrid combination of different numbers of layers of different graphene-related materials of different dimensions.

  Graphene-related materials can be combined with various auxiliary materials to design specific properties at the G-biointerface. Such properties include enhanced absorption efficiency, desired spectral activation profile, p- or n-type functionality, on-off switching speed, processability, durability, suitability, electrical conductivity, specificity Possible chemical functionalization and three-dimensional configurations to create biointerfaces of the. Examples of auxiliary materials include metals (eg, gold, silver, iron, iron oxide, titanium dioxide, lanthanide oxides, transition metals and transition metal oxides), graphene-like materials (eg, molybdenum disulfide, tungsten disulfide, Niobium selenide and boron nitride), semiconductors, silica, polymers or combinations thereof.

  Graphene-related materials can be combined with polymers, including but not limited to natural components of the extracellular matrix (eg, collagen, fibronectin and laminin) or synthetic polymers (eg, polyaniline, polypyrrole and polythiophene).

  The graphene-related material can be used as a stand-alone structure without any external structural support, or can be deposited directly on a substrate. They can also be combined with other materials that can provide a free standing support. The specific spatial arrangement of the G-biointerface can be achieved both by exploiting the unique dimensions of various graphene-related materials and by incorporating appropriate auxiliary materials.

  The external dimensions of the G-biointerface in the XY plane can range from nano-sized (eg, 1 nano-sized graphene flakes) to macro-sized, limited only by manufacturing capabilities. The minimum dimension of the XY plane is 1 nm × 1 nm. The outer dimensions along the Z-axis depend on the number of layers of graphene-related material incorporated into the G-biointerface. The minimum dimension along the Z axis is defined by the thickness of the non-functional graphene monolayer, 0.34 nm.

  The target can be one or more intact cells, one or more cell fractions, or one or more artificial membrane structures. Examples of cell fractionation include any luminal organelles, such as nuclei, ribosomes, mitochondria, endoplasmic reticulum, Golgi apparatus, vacuoles, synaptic vesicles, and lysosomes. Examples of artificial membrane structures include phospholipid micelles, micro- and nanocapsules and semi-fluid films on support structures.

  The one or more cells can generally be any type of cell having a membrane and a membrane potential. For example, the cell can be a bacterial (gram-positive or gram-negative), eukaryotic, prokaryotic, fungal, insect, bird, reptile, oocyte, fly, zebrafish, fish, nematode, amphibian or mammalian cell. The method may also be used on non-cellular materials such as artificial membranes, liposomes and phospholipid bilayers. Examples of primary mammalian cells include human, mouse, rat, dog, cat, bear, mousse, cow, horse, pig or Chinese hamster ovary ("CHO") cells. Other examples of cell types include immune system cells (eg, B cells, T cells), oocytes, red blood cells, white blood cells, neurons, CM, epithelium, glia, fibroblasts, stem cells, cancer cells, secretory cells Cells and immortalized cells.

  In order to be activated via the G-biointerface, cells must be placed in contact with or very close to the surface of the graphene-related material. To achieve such an arrangement, any cells can be added to the G-biointerface, or the G-biointerface can be added to the cells.

  After properly positioning the cells, the G-biointerface must be exposed to electromagnetic stimuli including, but not limited to, the visible spectrum, high frequency spectrum, infrared, microwave, terahertz frequencies or combinations thereof. For example, exposure of the G-biointerface to electromagnetic radiation (ie, light) in the visible spectrum can be performed by essentially any illumination method including, but not limited to, laser, mercury lamp, xenon lamp, halogen lamp illumination or LED illumination.

  Stimulation of cells via the G-biointerface may be combined with other means of stimulation, including but not limited to electrical stimulation, magnetic stimulation, chemical stimulation, biological stimulation, or combinations thereof.

  The G-biointerface can be used for studying the processes underlying cell differentiation, migration, proliferation and excitation-neurogenic coupling to guide activity-dependent maturation and differentiation of stem cell-derived neurons. In other embodiments, a G-biointerface may be utilized for the generation of stem cell-derived cells of a particular maturity. Such stem cell-derived cells can be patient-specific cells. Stem cell types include embryonic stem cells, adult stem cells and iPSCs. Examples of cells include, but are not limited to, neurons, CM, photoreceptors, fibroblasts, endothelial cells, pancreatic cells, muscle cells, secretory cells, adipocytes, mesenchymal stem cells and osteogenic stem cells.

  The G-biointerface also promotes functional recovery after nerve injury by relying on accelerated stimulatory stimulation of axonal outgrowth, thereby increasing reliance on nerve regeneration research and electrical and stimulation techniques. It can also be used to help develop neural function substitutes through integration with neural networks for the treatment of certain neurological disorders.

Remote stimulation of cells via the G-biointerface can be combined with optical methods for monitoring cell activity. The optical output signal can be captured using brightfield and / or fluorescent light microscopy, which can be the result of either label-free experiments or fluorescent labeling of the cells. Fluorescent dyes and / or proteins are involved in a variety of events in living cells that are activated by light, such as changes in intracellular calcium using Ca ²⁺ sensitive fluorescent indicators or changes in membrane potential using voltage sensitive fluorescent probes. Can be used for real-time imaging. Alternatively, the optical assay of G-biointerface stimulator cells can be an endpoint assay that analyzes fixed cells via immunohistochemical methods. The optical signal may generally be monitored using any suitable light measurement or light storage device. Examples of such devices include optical cameras, digital cameras, CCD cameras, CMOS cameras, video devices, CIT imaging, any digital camera mounted on a microscope, photomultiplier tubes, fluorimeters, luminometers, spectrophotometers. And even the human eye.

  Remote stimulation of cells via the G-biointerface can be combined with patch clamp techniques, planar electrophysiological systems, microelectrode arrays, field transistors and electrical recording of cell activity including but not limited to carbon nanotubes.

  In addition to providing stimulation of cells, if the biointerface can be used simultaneously to record cell activity using either imaging or electrical modalities or a combination thereof, the G-biointerface will Can be used for two-way communication.

  Remote stimulation of cells via the G-biointerface may include PCR, RT-PCR, DNA sequencing, next generation sequencing, DNA microarray, karyotyping, fluorescence in situ hybridization, calorimetric in situ hybridization or a combination thereof. It can be combined with their subsequent genetic analysis, including but not limited to.

  Cellular stimulation via the G-biointerface is a) study of synaptic plasticity in neuronal networks; b) sensations and manifestations in many neuropathies such as Parkinson's disease, Alzheimer's disease, autism and amyotrophic lateral sclerosis. Studies of the mechanisms underlying motor dysfunction; c) heart studies; d) monitoring of the functional activity of cells under study, including studies of pancreatic disorders.

  The G-biointerface can be utilized for pharmacological profiling of cells. In this case, the benchmark compound is tested in cells of unknown genetic makeup, while adjusting the cell membrane potential with G-biointerface electromagnetic radiation, and the cell response is monitored using any available detection method.

  The G-biointerface can be used for drug discovery screening applications, i.e., for pharmacological characterization of new chemicals. In this case, well-characterized cells in various activation states regulated by the G-biointerface are exposed to the novel chemical and the cell response is monitored using any available detection method. Importantly, by regulating the membrane potential of cells during drug screening, the G-biointerface may rely on efficient use, streamline the development of activity-dependent drugs, and thus reduce on-target and It may be beneficial for the characterization of both off-target effects as well as for detecting the potential toxicity of new compounds in various cell types in various activation states.

  The G-biointerface may be used to treat animals with medical conditions. Changes in cell membrane potential caused by G-biointerface electromagnetic radiation placed in close proximity to diseased cells and areas help to overcome the consequences of reduced or eliminated cell activity during disease progression. Examples of diseases that can be treated by the G-biointerface include, but are not limited to, arrhythmias, dysfunction after cardiac infraction, neurodegenerative / neurological disorders, various ophthalmological disorders and endocrine disorders.

  All of the compositions and / or methods and / or processes disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the present invention have been described with respect to preferred embodiments, it will be apparent to those skilled in the art that the compositions and / or compositions described herein can be prepared without departing from the spirit and scope of the present invention. Or, changes may be made to the method and / or process and steps or order of steps of the method. More specifically, it will be appreciated that the same or similar results may be achieved while replacing the agents described herein with certain agents that are both chemically and physically related. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the scope and concept of the invention.

Example 1: Graphene-related material Graphene was synthesized on a 25-μm-thick copper foil (Alpha Aesar, 13382, 99.8%) measuring 10 cm x 11 cm. Prior to the growth of graphene, the copper foil was cleaned by the following procedure. Immerse in a shallow acetone bath, perform mechanical cleaning in acetone, transfer to a similar bath filled with isopropyl alcohol (IPA), perform mechanical cleaning in IPA, dry in a stream of compressed air, and electrolytic polishing Then, it was rinsed with DI water and IPA, and blast-dried under a stream of compressed air. Atmospheric pressure CVD graphene synthesis was performed in a quartz tube furnace (MTI OTF-1200X-HVC-UL) with the following tube dimensions: d = 7.6 cm, l = 100 cm.

  In our experiments, we found that graphene oxide (eg, from Graphenea Inc.) and any chemical reduction of graphene oxide related materials (eg, hydrazine hydrate, sodium borohydride or L-ascorbic acid) ) Or reduced graphene oxide produced by thermal reduction was used. In some cases, thermal reduction was performed after chemical reduction.

  For a typical experiment to make rGO by chemical reduction, 1 mL of hydrazine hydrate was added to 100 mL of sonicated GO aqueous solution (1 mg / mL), and the resulting mixture was then added at 95 ° C. Incubation for 1 hour followed by filtration was included.

  In another exemplary experiment for making rGO by chemical reduction, 100 mg of L-ascorbic acid was added to 100 mL of sonicated GO aqueous solution (1 mg / mL) at room temperature, followed by vigorous over 48 hours. Agitation and centrifugation were included.

Example 2: Preparation of G-biointerface After cleaning glass coverslips (VWR) with Triton X-100 solution for 2 hours, they were thoroughly washed in ethanol and 2 hours under UV light. Let dry.

1. To initiate the transfer of graphene onto the glass coverslip, first apply poly (methyl methacrylate) (PMMA) in toluene at 4000 rpm for 60 seconds to a copper foil bearing a film of CVD grown graphene. Spin coating with a 5% w / w solution was performed. After spin coating, the exposed graphene on the copper foil side opposite to PMMA was etched using an oxygen plasma cleaner (30 s, 30 W, 200 mtorr oxygen pressure). Next, the PMMA / graphene-coated copper foil was floated in a 1 M iron (III) chloride (FeCl ₃ ) bath for 30 minutes to etch the copper. The floating PMMA-supported graphene was then transferred to a DI water bath three times, and then placed on a coverslip by "shooting" a free standing film from the DI water bath. After drying at room temperature for 2 hours, the coverslip was placed in an acetone bath overnight to remove PMMA. The coverslips were then rinsed in IPA and dried in compressed air.

  In a typical experiment, rGO made by chemical reduction of GO was placed on a glass coverslip by droplet deposition (10 μL / 12-mm coverslip). The rGO coverslips were then air dried for 30 minutes and then placed in a UV cell culture hood overnight for sterilization. Alternatively, we can: a) deposit GO droplets on a glass cover slip as described above, and then b) place them on a stage in an oven (T = 200 ° C.) for 5 minutes. An rGO coated coverslip was made by performing a direct thermal reduction of GO above.

  To make rGO coated 96-well microtiter plates, we used 20 mL of 0.1 mg / mL rGO solution prepared by chemical reduction using L-ascorbic acid in a standard 96-well microplate ( Deposited in each well of ThermoFisher Scientific). Next, the rGO coated plates were dried and sterilized overnight in a UV cell culture hood.

Example 3 Cell Culture Procedure iPSCs were generated by four factor programming and differentiated into NPCs. The NPC cultures are then placed on plastic dishes coated with 20 μg / mL poly-L-ornithine (Sigma) overnight, followed by 5 μg / mL laminin (Sigma) in an incubator for at least 2 hours, or 100 μg / mL. It was further coated with poly-L-ornithine and then placed directly on a graphene-coated glass coverslip coated with 10 μg / mL laminin. NMEM (Life Technologies), B27 (Life Technologies), Pen / Strep and 20 ng / mL fibroblast growth factor (FGF) (Millipore) supplemented with DMEM-F12 + Glutamax (LifeTechNolgie PC containing Negative Technologies containing PC). Maintained. The medium was changed every two or three days. When NSCs reached confluence, FGF was removed from the medium and maintained for 5 weeks with a medium change twice a week. After three weeks of differentiation, neurons were purified by FACS using the cell surface antigen signature. Differentiated neurons were detached using a 1: 1 mixture Accutase / Accumax (Innovative Cell Technologies) and stained with CD184, CD44 and CD24 antibodies (BD Biosciences). NPCs that were negatively stained for CD184 and CD44 and stained positively for CD24 were selected and NPC basal supplemented with 0.5 mM dbCAMP (Sigma), 20 ng / mL BDNF and 20 ng / mL GDNF (Peprotech). Seeded on poly-ornithine / laminin-treated graphene-coated cover slips in medium.

ICell CM (Cellular Dynamics International) was freeze-thawed according to the manufacturer's instructions. 25 μL of the cell suspension (160,000 cells / mL) was added to a gelatin-coated 384-well plate. The cells were then placed at 37 ° C. with 5% CO ₂ for 48 hours. Forty-eight hours later, 75 μL of chemically defined medium (CDM) was added to each well for a total of 100 μL, then half the volume was changed every other day. When CM started its spontaneous contraction after 10-14 days in culture, they were lifted with trypsin and replated at a density of 500,000 cells / 12-mm coverslips. CM was cultured on coverslips for 2-3 weeks before the experiment.

Example 4: Biocompatibility of CM and G-biointerfaces For biocompatibility assessment, we determined the morphology of cells cultured on glass coverslips with and without coating with graphene-related materials, Cell density and membrane integrity were compared. Bright field optical microscopy images show that CM shows normal morphology when cultured on glass coverslips coated with graphene and rGO (FIG. 2A). To gain further insight, we followed in scanning electron microscope (SEM) experiments. For these experiments, the CM was washed with 0.1 M phosphate buffer (pH 7.4), fixed with 4% formaldehyde solution for 2 hours at room temperature, and washed 3 times with the same buffer for 5 minutes each. did. Stepwise series of alcohols (50% ethanol-10 minutes, 70% ethanol-10 minutes, 80% ethanol-10 minutes, 95% ethanol-2 changes, 10 minutes, 100% ethanol-3 changes, 15 minutes) After dehydration, all samples were lyophilized in a vacuum chamber and coated with sputtered iridium. We obtained a scanning electron microscope image on an XL30 ESEM-FEG (FEI) at an imaging distance of 10 mm using a 10 kV energy beam. SEM images show that CM is in good agreement with both graphene and rGO coated glass substrates (FIG. 2B).

  To assess cell density, we calculated the number of cells in a predetermined area using bright-field light microscopy images (FIG. 3A). We have determined that CM prefers surfaces coated with graphene and rGO. FIG. 3B (left) shows that cell density was significantly higher on rGO coated glass coverslips than on “naked” glass coverslips.

  To assess cell viability, we used LIVE / DEAD® containing membrane permeable calcein-AM and ethidium homodimer-1 (EthD-1) impermeable to the membrane. The Viability / Cytotoxicity Kit (Life Technologies) was used. When stained with this kit, intact cell membranes and live cells with high levels of enzymatic activity appear green due to enzymatic conversion of non-fluorescent calcein-AM to green calcein and rejection of EthD-1. In contrast, EthD-1 only penetrates dead cells with damaged membranes, producing a red fluorescent signal upon binding to nucleic acids. Images of labeled cells were randomized using an Olympus I # 71 fluorescence microscope equipped with standard filters (fluorescein (for calcein), rhodamine (for EthD-1) and DAPI (for Hoechst)) and a QIClick CCD camera (QImaging Inc.). Taken on. Data was analyzed using ImageJ image analysis software. Cell death was calculated as a percentage ratio of the number of EthD-1 positive cells divided by the total number of EthD-1 positive and calcein positive cells. We determined that membrane integrity was not statistically different in cells cultured on coated versus "naked" substrates (FIG. 3B, right).

  To test whether culturing CM on the G-biointerface is beneficial for their functional activity, we used bright-field light microscopy and used a label-free approach. To monitor the spontaneous contraction of the CM. To detect the conformational changes that occur in a CM during CM contraction, we used a CMV to ensure that the luminosity inside the ROI changes as a result of spontaneous contraction. Just outside the end of the selected. Frequency was quantified by calculating the number of contractions per second. The present inventors have found that the frequency of CM contraction was higher when cells were cultured on the G-biointerface.

Example 5: G-biointerface and light-induced effects in CM Using the label-free approach described in Example 4, we determined that G-biointerface and spontaneous contraction of CM in two experimental configurations. The effects of biointerface light irradiation were studied: a) "long-term" (or chronic) harmonization when CM was cultured on rGO coated glass coverslips for 2-3 weeks and b) Control (e.g., non- "Coating""shortterm" (or acute) harmony when rGO slices were deposited on CM grown on coverslips for 10 minutes. The present inventors have found that light irradiation increases the frequency of CM contractions in both chronic (FIGS. 4A, B) and acute (FIG. 4C) experimental configurations.

Using an electrophysiological approach, we monitored the photo-induced changes in membrane potential of cells cultured on the surface of a glass coverslip coated with a graphene-related material. In a typical experiment, (mM _{in) NaCl, 135; KCl, 2.5} ; CaCl 2, 2; NaHCO 3, 1; Na 2 HPO 4, 0.34; KH 2 PO 4, 0.44; glucose, The rGO coated coverslip was placed in a laboratory chamber filled with an electrophysiological extracellular solution consisting of 20; HEPES, 10; and glycine, 0.01 (pH 7.4). The final chip resistors patch pipette 3~6MΩ, (in mM) CsCl, 120; tetraethylammonium _{chloride, 20; HEPES, 10; EGTA} , 2.25; CaCl 2, 1; MgCl 2, 2 (pH7.4) Was filled. All recordings were obtained using a Digidata 1322 interface, Axopatch 200B amplifier and pClamp software (Molecular Devices Corp.). We determined that light irradiation did not affect CM cultured on “naked” coverslips (FIG. 5A), but did cause membrane depolarization in CM on rGO-coated coverslips, which It was determined that this led to the start of a series of action potentials (FIG. 5B) or an increase in the frequency of action potential generation (FIG. 5C).

The all-optical assay combines photo-induced activation of cells (eg, via a G-biointerface) and optical methods to monitor cell activity (eg, using a fluorescent biosensor). In a typical experiment, the present inventors found that in a CO ₂ incubator, loading buffer (132mM NaCl; 4.2mM KCl; 1.8mM CaCl 2, 5mM D- glucose, 10 mM HEPES; adjusted to pH7.4) CM was loaded with either 5 μM Fluo-4 AM, a calcium-sensitive fluorescent indicator (Thermo Fisher Scientific) or 2 μM VF _1.2 , a voltage-sensitive dye for 45 minutes in the medium. To perform an all-optical assay, we used a short pulse of high intensity light at 520 nm to activate the G-biointerface and continuously illuminate with low intensity light at 480 nm to generate fluorescent light. The dye was excited. Imaging experiments were performed on an upright fluorescent microscope with a fluorescein filter set (excitation 480/20 nm, emission 525/30 nm). Images were obtained with a CCD camera at 30 Hz for a few seconds and analyzed using ImageJ software. In our experiments, we detected changes in intracellular calcium concentration resulting from the generation of photo-evoked action potentials in CM (FIG. 6), and The feasibility of an all-optical assay incorporating a G-biointerface was confirmed.

Example 6: Biocompatibility of neurons and G-biointerface We used both brightfield light microscopy and SEM to morph the iPSC-derived neurons cultured on glass coverslips coated with graphene-related materials Was evaluated (FIG. 7). We determined that iPSC-derived neurons did not change their morphology after 6 weeks of culture on rGO coated glass coverslips.

  To evaluate the biocompatibility of iPSC-derived neurons and rGO coated glass cover slips, we performed viability experiments using the LIVE / DEAD® Viability / Cytotoxicity Kit (Life Technologies). We analyzed the results as described in Example 4 and determined that cell viability was higher on rGO coatings than on "naked" glass coverslips (FIG. 8).

Example 7: G-biointerface and light-induced effects on neurons We performed electrophysiological studies on iPSC-derived neurons according to the experimental protocol described in Example 5 (FIG. 9A). The inventors determined that, as in CM, light irradiation caused membrane depolarization in neurons, which led to the generation of action potentials (FIG. 9B).

  To perform an all-optical check of neuronal activity, we illuminated their rGO-coated substrates and described them using Fluo-4, a fluorescent calcium-sensitive indicator, as described in Example 6. The neurons were activated by monitoring the light-induced activity of (Fig. 10A). We successfully recorded changes in intracellular calcium during electrical activation induced by light irradiation only in neurons cultured on rGO coated substrates (FIG. 10B).

Example 8: Statistical analysis Data are means ± sd. e. m. Expressed as Where appropriate, one-way ANOVA with Student-Newman-Keuls (SNK) post-test was used to interpret the results. Pairwise comparisons between genotypes / treatments were evaluated by Student's t-test. Differences were considered statistically significant at P <0.05.

  In the above detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be used and other modifications may be made without departing from the spirit or scope of the subject matter provided herein. Aspects of the present disclosure may be arranged, substituted, combined, separated, designed in a wide variety of different arrangements, as generally described herein and illustrated in the drawings, all of which are incorporated herein by reference. It will be readily understood that it is explicitly contemplated herein.

  This disclosure is not limited with respect to the particular embodiments described herein, which are intended to be illustrative of the various aspects. As will be apparent to those skilled in the art, many modifications and variations can be made without departing from the spirit and scope of the invention. Functionally equivalent methods and apparatuses within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to be included within the scope of the appended claims. The present disclosure, together with the full scope of equivalents to which such claims are entitled, is limited only by the terms of the appended claims. It is to be understood that this disclosure is not limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not limiting.

  As used in this document, the singular forms “a”, “an”, and “the” mean the plural unless the context clearly dictates otherwise. including. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Nothing in this disclosure should be construed as an admission that the embodiments described in this disclosure do not precede such disclosure by virtue of prior invention. As used in this document, the term "comprising" means "including, but not limited to".

  While the term "comprising" describes various compositions, methods and devices, it refers to the various components or steps ("including, but not limited to"). Compositions, methods and devices may also be "consistent essential of" the various components and steps, or may be "consistent of" Terms should be construed as essentially defining a limited set of elements.

  With respect to the use of substantially any plural and / or singular terms herein, those skilled in the art may convert plural to singular and / or singular to plural as appropriate to the content and / or application. . Various singular / multiple changes may be explicitly shown for clarity.

  In general, terms used herein and in particular in the appended claims (eg, the text of the appended claims) are generally intended to be “non-limiting” terms (eg, “ The term "including" is to be interpreted as "including, but not limited to" and the term "having" is interpreted as "at least having". It should be understood by those skilled in the art that the term "includes" should be interpreted as "includes, but is not limited to". Where the specific number of recitations of claims to be introduced is intended, such intent is explicitly stated in the claims, and without such intent, it will be further understood by those skilled in the art that such intent does not exist. . For example, as an aid to understanding, the following appended claims may contain the use of the prefixes "at least one" and "one or more" to introduce claim recitations. However, the use of such a phrase means that the same claim may require that the introductory phrases “one or more” or “at least one” and indefinite articles such as “one (a)” or “one (an)” (eg “ "A" and / or "an" should be taken to mean "at least one" or "one or more"), Introducing claims in the definite article "one (a)" or "one (an)", for embodiments containing only one such claim, such claims It should not be construed as suggesting a limitation on any particular claim containing a claim, and the same applies to the use of definite articles used to introduce claim claims. Furthermore, even if the specific number of claims to be introduced is explicitly stated, one of ordinary skill in the art should interpret such description to at least mean the recited number (eg, , A simple description of “two statements” without other modifiers means at least two statements or more than one statement). Further, in instances where similar notation is used, such as "at least one of A, B, and C, etc.", such syntax is generally intended in the sense that one of ordinary skill in the art would understand this notation. (Eg, “a system having at least one of A, B and C” is A only, B only, C only, A and B together, A and C together, B and C are Including but not limited to systems having A and B and C together and / or the like). In instances where notations similar to "at least one of A, B or C etc." are used, such syntax is generally intended in the sense that one of ordinary skill in the art would understand the notation (eg, , "A system having at least one of A, B or C" refers to A only, B only, C only, A and B together, A and C together, B and C together and / or A , B and C together, and the like). Disjunctive terms and / or phrases that provide virtually any two or more alternative terms, whether in this specification, in the claims or in the drawings, are one of those terms, any of the terms It will be further understood by those skilled in the art that it is intended to include the possibility of including both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

  Furthermore, where features or aspects of the disclosure are described in a Markush group, those of skill in the art will recognize that the disclosure is also thereby described in terms of any individual element or subgroup of elements of the Markush group. .

  As will be understood by those skilled in the art, for any purpose, such as in connection with providing this specification, all ranges disclosed herein are inclusive of all possible sub-ranges and sub-ranges thereof. Combinations are also included. Fully explain that any listed range is at least equally divided into two, three, four, five, ten, etc. Can be easily recognized as By way of non-limiting example, each range discussed herein can be readily divided into a lower third, a middle third, a higher third, and so on. As will also be appreciated by those of skill in the art, languages such as "at a maximum", "at least", and the like, all include the recited numbers, and refer to ranges that are separated by subranges as discussed above. Finally, as will be understood by those skilled in the art, a range includes each individual element. Thus, for example, a group having 1-3 cells refers to groups having 1, 2 or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and the like.

  Various of the above disclosed and other features and functions, or alternatives thereof, may be combined in many other different systems or applications. Various currently unforeseen or unexpected alternatives, modifications, variations or improvements therein may be subsequently made by those skilled in the art, each of which is intended to be encompassed by the disclosed embodiments.

Claims (27)

At least one biointerface,
A system for regulating an activation state of a cell, comprising at least one source of electromagnetic radiation and at least one biological target, the system comprising :
The biointerface is configured to generate free charge carriers in response to exposure to electromagnetic radiation, the biointerface comprising graphite; graphene; polycyclic aromatic hydrocarbons; carboxyl groups, esters, amides, thiols, Functionalized graphite, graphene, functionalized with one or more of hydroxyl, diol, ketone, sulfonate, carbonyl, aryl, epoxy, phenol, phosphonic, amine, porphyrin, pyridine, polymer Polycyclic aromatic hydrocarbons; graphite, graphene, polycyclic aromatic hydrocarbons in which carbon atoms are partially substituted with non-carbon atoms; graphite oxide; graphite oxide containing an oxygen-containing functional group; reduced graphene oxide; Some oxygen-containing functional groups Comprising one or more materials selected from the group consisting of:
The electromagnetic radiation source is configured to expose at least one biointerface to electromagnetic radiation;
The biological target contacts the at least one biointerface such that free charge carriers generated by exposure of the at least one biointerface to electromagnetic radiation remotely and reversibly manipulate the cell membrane potential of living cells. Or a system for regulating the activation state of cells, including living cells placed in close proximity. System for adjusting the one or more materials, graphite, graphene, graphite oxide, graphene oxide of claim 1 comprising a reduced form graphene oxide or a combination thereof, the activation state of the cells. The system for regulating the activation state of a cell according to claim 1, wherein the at least one bio-interface is a hybrid structure of one or more materials combined with a combination of one or more auxiliary materials. The auxiliary material is gold, silver, platinum, cobalt, copper, iron, silicon, germanium, transition metal, iron oxide, titanium dioxide, lanthanide oxide, transition metal oxide, perovskite, silica, porous silica, semiconductor, crystal. The structure according to claim 3, wherein the structure is composed of a non-crystalline, amorphous, layered material, protein, polymer, inorganic core with polymer shell, organic or inorganic core with polymer shell and inorganic core with polymer shell, or a combination thereof. , A system that regulates the activation state of cells . The auxiliary material is collagen, fibronectin, vitronectin, osteocalcin, laminin, cadherin, intaglin, lectin, nectin, claudin, Ig superfamily cell adhesion molecule, RGD cell adhesion peptide, polysaccharide collagen (gelatin), alginate, cellulose. , Starch, chitosan (chitin), fibrin, albumin, gluten, elastin, fibroin, hyaluronic acid, sclerolcan, ercinan, pectin (pectic acid), galactan, curdlan, gellan, leban, emulsan, dextran, pullulan, heparin, silk, chondroitin 6-sulfate, according to claim 3 which is a natural polymer comprising a polyhydroxyalkanoate or a combination thereof, systems which regulate the activation status of the cells . The auxiliary material may be poly (lysine), poly (ornithine), poly (ethyleneimine), poly (valine), poly (aniline), poly (N-isopropylacrylamide), poly (lactide), poly (thiophene), poly (thiophene) (Vinyl alcohol), poly (urethane acrylate), poly (N-methylpyrrole), poly (vinylpyrrolidone), poly (pyrrole), polysilane, poly (ethylene glycol), poly (Y-benzyl-L-glutamate) -block Copolymer, 2-methacryloyloxyethyl phosphorylcholine polymer, poly (p-xylylene), poly (N-isopropylacrylamide), poly (caprolactone), polyhydroxybutyrate), poly (propylene fumarate), α-hydroxyester, poly (Anhydride), poly ( Sphazene), poly (phosphoester), hydrogel, poly (glutamic acid), poly (glycolic acid), poly (lactic acid) and copolymers thereof, poly (styrenesulfonate), sulfonated poly (ether-etherketone), poly (sulfone) ), Poly (diallyldimethylammonium chloride), poly (octadecylsiloxane), poly (styrene block-poly-4-vinylpyridine), poly (methyl methacrylate), poly (hexyl methacrylate), poly (methacrylic acid), poly (N-methacrylic acid) The system for regulating the activation state of a cell according to claim 3, which is a synthetic polymer containing N, N'-dimethylaniline), poly (trans-3- (3-pyridyl) acrylic acid) or a combination thereof. The system for regulating the activation state of a cell according to claim 1, wherein the one or more materials are arranged in a continuous, spot-like, stripe-like pattern, or a combination thereof. The system for regulating the activation state of a cell according to claim 1, wherein the at least one biointerface is a free-standing structure without an external structural support. The at least one bias interface further comprises a structural material selected from the group consisting of a glass material, a metal material, a ceramic material, a semiconductor material, a dielectric material, a plastic material, a polymer material, a layer of adhesive molecules, or a combination thereof. The system for regulating the activation state of a cell according to claim 1. The system for regulating the activation state of a cell according to claim 1, wherein the at least one biointerface is located in a well of a microtiter plate. Providing at least one biological target,
Disposing the at least one biological target and at least one biointerface configured to generate free charge carriers in response to exposure to electromagnetic radiation;
The biointerface is graphite; graphene; polycyclic aromatic hydrocarbon; carboxyl group, ester, amide, thiol, hydroxyl group, diol group, ketone group, sulfonate group, carbonyl group, aryl group, epoxy group, phenol group, phosphone group. Functionalized graphite, graphene, polycyclic aromatic hydrocarbons functionalized with one or more of acids, amine groups, porphyrins, pyridines, polymers; graphite, graphene, in which some of the carbon atoms are replaced by non-carbon atoms Including one or more materials selected from the group consisting of polycyclic aromatic hydrocarbons; graphite oxide; graphite oxide containing oxygen-containing functional groups; reduced graphene oxide; graphene oxide lacking at least some oxygen-containing functional groups. ,
The biological target contacts the at least one biointerface such that free charge carriers generated by exposure of the at least one biointerface to electromagnetic radiation remotely and reversibly manipulate the cell membrane potential of living cells. Or including live cells that are placed in close enough proximity,
A method for inducing a change in the functional state of a biological target in vitro, comprising exposing said at least one bioion interface to electromagnetic radiation.   Exposure of said at least one baryon interface to electromagnetic radiation is exposure to visible spectrum electromagnetic radiation, high frequency spectrum electromagnetic radiation, infrared electromagnetic radiation, terahertz frequency spectrum electromagnetic radiation, microwave spectrum electromagnetic radiation, or a combination thereof. The method according to claim 11.   The method of claim 11, wherein exposing the at least one vion interface to electromagnetic radiation is exposure to visible light. 12. The method of claim 11, comprising utilizing the at least one biointerface for stem cell research and generation of stem cell-derived cells of a particular maturity .   12. The method of claim 11, wherein the at least one biointerface is used for electroporation of a biological membrane and for delivery of an electroporation-based substance through the biological membrane. Providing at least one biological target, including living cells,
Positioning live cells of said at least one biological target and at least one biointerface to generate free charge carriers in response to exposure to electromagnetic radiation;
The living cells are contacted or sufficiently contacted with at least one biointerface such that free charge carriers generated by exposure of the at least one biointerface to electromagnetic radiation remotely and reversibly manipulate the cell membrane potential of the living cells. Placed in close proximity,
The biointerface includes graphite; graphene; polycyclic aromatic hydrocarbon; carboxyl group, ester, amide, thiol, hydroxyl group, diol group, ketone group, sulfonate group, carbonyl group, aryl group, epoxy group, phenol group, phosphone group. Functionalized graphite, graphene, polycyclic aromatic hydrocarbons functionalized with one or more of acids, amine groups, porphyrins, pyridines, polymers; graphite, graphene, in which some of the carbon atoms are replaced by non-carbon atoms Including one or more materials selected from the group consisting of polycyclic aromatic hydrocarbons; graphite oxide; graphite oxide containing oxygen-containing functional groups; reduced graphene oxide; graphene oxide lacking at least some oxygen-containing functional groups. ,
Activating the at least one biointerface by exposing the at least one biointerface to electromagnetic radiation;
Elicit a change in the functional state of at least one biological target through manipulating the cell membrane potential;
Monitoring the resulting change in said at least one biological target using one or more detection methods,
A method for performing an in vitro biological assay wherein said target is one or more intact cells, one or more cell fractions or one or more artificial membrane structures.   17. The method of claim 16, wherein monitoring of the at least one biological target is performed using an optical detection method.   Monitoring at least one biological target is monitoring the functional status of the target, the change being morphology, cell migration, cell viability, cell metabolism, intracellular ion concentration, cell membrane potential, mitochondrial membrane potential. 17. The method of claim 16, comprising altering one or more of intracellular / extracellular concentrations of signaling molecules (eg, glutamate, cAMP), enzymatic activity, translocation of intracellular molecules, or a combination thereof.   17. The monitoring of the biological target using an electrophysiological detection method including patch clamp techniques, planar electrophysiological systems, extracellular recordings, microelectrode arrays, field transistors, carbon nanotubes or a combination thereof. The method described in.   The monitoring of the at least one biological target comprises PCR, RT-PCR, DNA sequencing, next generation sequencing, DNA microarray, karyotyping, fluorescence in situ hybridization, calorimetric in situ hybridization, or a combination thereof. 17. The method according to claim 16, wherein the method is performed using a genetic detection method.   By stimulating at least one biological target and monitoring a resulting change in the biological target using at least one biointerface, either in an imaging or electrical fashion, or a combination thereof; 17. The method of claim 16, further comprising utilizing for at least one biological target for two-way communication. 17. The method according to claim 16, which is used for evaluating a chemical substance with respect to a pharmacological property, a potential cardiotoxicity, a neurotoxicity, a metabolic toxicity or a combination thereof , or for screening a chemical substance or pharmacological profiling. The described method.   17. The method of claim 16, wherein the method is used for pharmacological profiling of cells, membrane receptors, ion channels, transmembrane transporters, kinases, phosphatases, or combinations thereof. A system for treating an animal having a medical condition , comprising:
At least one biointerface configured to generate free charge carriers in response to exposure to electromagnetic radiation, and at least one source of electromagnetic radiation configured to expose the at least one biointerface to electromagnetic radiation ,
The biointerface includes graphite; graphene; polycyclic aromatic hydrocarbon; carboxyl group, ester, amide, thiol, hydroxyl group, diol group, ketone group, sulfonate group, carbonyl group, aryl group, epoxy group, phenol group, phosphone group. Functionalized graphite, graphene, polycyclic aromatic hydrocarbons functionalized with one or more of acids, amine groups, porphyrins, pyridines, polymers; graphite, graphene, in which some of the carbon atoms are replaced by non-carbon atoms Including one or more materials selected from the group consisting of polycyclic aromatic hydrocarbons; graphite oxide; graphite oxide containing oxygen-containing functional groups; reduced graphene oxide; graphene oxide lacking at least some oxygen-containing functional groups. ,
At least one organism containing living cells is administered such that free charge carriers generated by exposure to electromagnetic radiation of the at least one biointerface upon administration to the animal remotely and reversibly manipulate the cell membrane potential of the living cells. A biological target is placed in contact with or in close proximity to at least one biointerface;
The medical condition is Alzheimer's disease; Parkinson's disease; Amyotrophic lateral sclerosis; Huntington's disease; Chemotherapy-induced neuropathy; Age-related cerebral dysfunction; Down's syndrome; Autism spectrum disorder; Cerebral palsy; Epilepsy; Disorders; emotional disorders, depression, traumatic brain injury, post-traumatic stress disorder (chronic traumatic brain injury), stressful, traumatic or blast damage or disease; schizophrenia and other mental disorders; pain, hyperalgesia and Nociceptive disorders; Addiction disorders; Nerve ischemia; Nerve reperfusion injury; Nerve trauma; Nerve hemorrhage; Nerve injury; Nerve infection; Stroke; Cancer; Cardiovascular disorders; Arrhythmias; Heart failure; Cardiomyopathy; Rheumatic heart disease Coronary artery disease; congenital heart disease; eye disease; age-related macular degeneration; retinitis pigmentosa; glaucoma: diabetic retinopathy; calcium homeostasis disorder; glucagon It is sexual dysfunction; live tumor; diabetes; hypoglycemia; adrenal disorders; Thyroid disorders; growth disorders; metabolic bone disease; hormone disorders;
A system for treating or preventing an animal having a medical condition, wherein the animal is a mammal, human, primate, dog, cat, mouse, rat, cow, horse or pig.   25. The system of claim 24, comprising utilizing at least one biointerface for a neural function substitute.   25. The system of claim 24, comprising utilizing at least one biointerface for a retinal implant.   25. The system of claim 24, comprising utilizing at least one biointerface for combination with stem cell replacement therapy.

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